Recent work has resulted in a simplified approach to the isolation of highly purified C5a from a purified C5 as well as citrated plasma. Specific antibody staining of nitrocellulose containing C5a electrophoretically transferred from SDS-PAGE gels confirms the presence, size and purity of this C5 cleavage fragment. Human C5a prepared by this method was shown to be a potent chemoattractant for neutrophils and monocytes, and an aggregator of neutrophils. C5a prepared from purified human C5 with zymosan bound alternate pathway convertase was used for in vivo skin test studies in normal volunteers. These studies revealed that C5a produced immediate wheal and flare reactions in all individuals, and was active in doses as low as 1 ng 10 to to the - 13 mole). A clear dose-response effect was obtained between 1 and 120 ng. Comparison of in vivo reactivity of human C5a with C3a, histamine, 48/80, and morphine sulfate demonstrated that C5a was the most potent mediator of wheal and flare reactions. In addition several pharmacologic agents commonly employed to treat dermatologic and/or allergic disorders were tested for their ability to modulate cutaneous reactivity to C5a. This study is the first detailed analysis of the cutaneous reaction pattern to C5a in man and demonstrates that C5a is a potent mediator of inflamation in human skin in vivo. C5a generation in vivo by cell bound classical pathway convertase (EAC1423) was examined in relation to the terminal complement components C6-9. It was shown that the presence of terminal components during activation of C5 allowed generation of significantly greater levels of C5a antigen and biological activity although consumption of C5 substrate was similar whether or not the terminal components were present. C5a antigen detected by radioimmunoassay correlated with C5a biological activity assessed by polymorphonuclear myeloperoxidase release and thus describes a new role for the human terminal complement components in generation of biologically active C5a.